The purified products of the bacteriophage T4 genes 32, 41, 43, 44, 45, 61, & 62 together catalyze extensive DNA synthesis on single and double-strand DNA templates by a mechanism closely approximating in vivo DNA replication process. We have developed a sensitive assay for the determination of the accuracy of DNA replication in vitro as catalyzed by these proteins. Using this assay we are examining the roles played by the various factors involved in this reaction (salts, divalent cation, nucleotide concentrations and the various proteins) on the fidelity of replication. We are also using this in vitro system to identify the sites of initiation of DNA synthesis on single- and double-stranded DNA templates. The initiation sites on a number of different templates will be compared to try to identify the features in the DNA sequence that play significant role in the initiation process. Finally, we are using this in vitro DNA replication apparatus to examine the effects of carcinogen damage to DNA on the template activity of this molecule. We shall attempt to understand the nature of alterations that lead to mutagenesis during in vitro DNA replication.